Foreign Cry1ac Gene Integration and Endogenous Borer Stress-Related
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Zhou et al. BMC Plant Biology (2018) 18:342 https://doi.org/10.1186/s12870-018-1536-6 RESEARCHARTICLE Open Access Foreign cry1Ac gene integration and endogenous borer stress-related genes synergistically improve insect resistance in sugarcane Dinggang Zhou1,2†, Xiaolan Liu1,2†, Shiwu Gao1, Jinlong Guo1, Yachun Su1, Hui Ling1, Chunfeng Wang1, Zhu Li1, Liping Xu1* and Youxiong Que1* Abstract Background: Sugarcane (Saccharum spp. hybrids) is considered the most globally important sugar-producing crop and raw material for biofuel. Insect attack is a major issue in sugarcane cultivation, resulting in yield losses and sucrose content reductions. Stem borer (Diatraea saccharalis F.) causes serious yield losses in sugarcane worldwide. However, insect-resistant germplasms for sugarcane are not available in any collections all over the world, and the molecular mechanism of insect resistance has not been elucidated. In this study, cry1Ac transgenic sugarcane lines were obtained and the biological characteristics and transgene dosage effect were investigated and a global exploration of gene expression by transcriptome analysis was performed. Results: The transgene copies of foreign cry1Ac were variable and random. The correlation between the cry1Ac protein and cry1Ac gene copies differed between the transgenic lines from FN15 and ROC22. The medium copy lines from FN15 showed a significant linear relationship, while ROC22 showed no definite dosage effect. The transgenic lines with medium copies of cry1Ac showed an elite phenotype. Transcriptome analysis by RNA sequencing indicated that up/down regulated differentially expressed genes were abundant among the cry1Ac sugarcane lines and the receptor variety. Foreign cry1Ac gene and endogenous borer stress-related genes may have a synergistic effect. Three lines, namely, A1, A5, and A6, were selected for their excellent stem borer resistance and phenotypic traits and are expected to be used directly as cultivars or crossing parents for sugarcane borer resistance breeding. Conclusions: Cry1Ac gene integration dramatically improved sugarcane insect resistance. The elite transgenic offspring contained medium transgene copies. Foreign cry1Ac gene integration and endogenous borer stress- related genes may have a synergistic effect on sugarcane insect resistance improvement. Keywords: Sugarcane, cry1Ac gene, Insect resistance, RNA-Seq, Transcriptome analysis, Phenotypic traits * Correspondence: [email protected]; [email protected] †Dinggang Zhou and Xiaolan Liu contributed equally to this work. 1Key Laboratory of Sugarcane Biology and Genetic Breeding, Fujian Agriculture and Forestry University, Ministry of Agriculture, Fuzhou 350002, Fujian, China Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhou et al. BMC Plant Biology (2018) 18:342 Page 2 of 14 Background number of the foreign cry1Ac gene on its expression Sugarcane (Saccharum spp. hybrids) is considered the level in transgenic sugarcane via particle bombardment, most important crop for sugar production globally and is apart from the findings of Joyce et al. [23], who sug- a valued raw material for the biofuel industry [1, 2]. Insect gested that transgene copy number does not influence attack is a major issue in sugarcane cultivation, resulting the gene expression in transgene lines. in yield losses and sucrose content reduction [2]. One sig- The objective of breeding is to obtain excellent agro- nificant sugarcane pest is stem borer (Diatraea saccharalis nomic characters or to retain the agronomic characteris- F., Lepidoptera, Crambridae), which affects sugarcane tics of the parental cultivar [24]. Thus, assessing the throughout the entire growing season and causes serious agronomic performance of transgenic sugarcane under yield losses of nearly 25–30% [3, 4]. However, insect-resist- field conditions is necessary in order to select excellent ant sugarcane germplasms are not available in any collec- transgenic offspring [23]. Arencibia et al. [25]werethe tions [3]. In addition, modern sugarcane cultivars are first to perform field trials of five insect-resistant trans- highly complex polyploid-aneuploids, with chromosome genic sugarcane events and they found that most of the numbers ranging from 80 to 130 [5]. It is thus almost im- transgenic lines had agronomic traits similar to that of the possible to breed an insect-resistant variety by means of receptor variety. Gao et al. [18] tested the field perform- traditional cross-breeding. Genetic engineering is ex- ance of transgenic cry1Ac sugarcane and discovered that pected to play an important role in improving the insect these lines exhibited better phenotypic traits than the resistance of sugarcane [6–8] and could facilitate the de- non-transgenic sugarcane. Wang et al. [19] investigated velopment of insect-resistant sugarcane varieties or germ- the field performance of five single-copy transgenic sugar- plasms for use in cross-breeding. cane lines, which exhibited excellent cane borer resistance To date, the cry gene has been effectively used to con- and herbicide tolerance but poor agronomic traits. trol stem-borer pests in many crops, including rice The genetic engineering of plants with enhanced toler- (Oryza sativa)[9], corn (Zea mays)[10], cotton (Gossy- ance to biotic stresses (i.e., insect stress) typically in- pium hirsutum)[11], potato (Solanum tuberosum)[12] volves complex multigene networks and may therefore and soybean (Glycine max)[13], which have widespread have the potential to introduce unintended effects due commercial applications and verified safety [14–16]. to the location effect of transgene integration [26]. How- There is an urgent need for borer resistance traits, and a ever, the cry1Ac transgenic sugarcane insertion site and series of studies that introduced the cry gene into sugar- its flanking sequence are complex, and the effects of par- cane successfully obtained insect-resistant sugarcane ticle bombardment remain unclear. The growth and de- lines [1, 4, 17–19]. However, only one case involving velopment of plants, including transgenic plants, along insect-resistant transgenic sugarcane has been approved with plant responses to abiotic and biotic stresses, in- for commercial planting in Brazil. The first report on volves a complex network of gene regulation [27]. In re- insect-resistant sugarcane lines based on the use of the cent years, there has been an increasing number of cry1Ab gene [20]. Recently, Gao et al. [18] successfully reports on gene expression analysis in plants during de- introduced the cry1Ac gene into sugarcane and obtained velopment and stress using a global transcriptomic ap- insect- resistant transgenic lines. Wang et al. [19] suc- proach [27]. However, these reports mainly focused on cessfully introduced the cry1Ab and EPSPS genes to- model plants due to their available genome sequence in- gether and obtained transgenic sugarcane lines with formation. Many studies have investigated the transcrip- insect resistance and herbicide tolerance. tomes of non-model plants, including sugarcane, for The influence of transgene copy number on gene ex- which the sequencing of the whole genome is currently pression levels is complex [21]. The gene balance hy- in progress [27]. Microarrays, serial analysis of gene pothesis suggests that increasing the transgene copy expression and RNA sequencing (RNA-Seq) are the number would upregulate gene expression levels, and three most commonly used methods for transcriptome thus a correlation (positive or negative) must exist be- analysis in gene expression studies [28, 29]. RNA-Seq, tween gene copy number and gene expression level [22]. which allows for the near-complete characterization of Therefore, transcript abundance must increase with gene transcriptomic events occurring in a specific tissue at a dosage in order to increase protein abundance [22]. certain time, has been widely applied and proven par- However, low copy-number exogenous genes are consid- ticularly useful in non-model plants, including sugarcane ered to be beneficial for plant improvement, particularly [28, 30–32]. For transgenic plants, a number of studies in diploid plants [21]. It is generally considered that a to date have used transcriptome analysis to study gene low gene copy number would decrease the possibility of expression or assess the impact of genetic engineering transgene co-suppression, while multiple gene copies [29, 33–36]. Misra et al. [35] identified differentially may result in gene silencing and co-suppression [21]. To expressed genes (DEGs) in AtMYB12-expressing trans- date, very little is known about the influence of the copy genic tobacco lines by microarray transcriptome analysis. Zhou et al. BMC Plant Biology (2018) 18:342 Page 3 of 14 Nietzsche et al. [36] employed a global transcriptional The CYC gene was selected for transgene copies esti- profiling approach using microarray to identify